Caveolin transfection results in caveolae formation but not apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins in epithelial cells.
Most epithelial cells sort glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface. The "raft" hypothesis, based on data mainly obtained in the prototype cell line MDCK, postulates that apical sorting depends on the incorporation of apical proteins into cholesterol/glycosphingolipid (GSL) rafts, rich in the cholesterol binding protein caveolin/VIP21, in the Golgi apparatus. Fischer rat thyroid (FRT) cells constitute an ideal model to test this hypothesis, since they missort both endogenous and transfected GPI-anchored proteins to the basolateral plasma membrane and fail to incorporate them into cholesterol/glycosphingolipid clusters. Because FRT cells lack caveolin, a major component of the caveolar coat that has been proposed to have a role in apical sorting of GPI-anchored proteins (Zurzolo, C., W. Van't Hoff, G. van Meer, and E. Rodriguez-Boulan. 1994. EMBO [Eur. Mol. Biol. Organ.] J. 13:42-53.), we carried out experiments to determine whether the lack of caveolin accounted for the sorting/clustering defect of GPI-anchored proteins. We report here that FRT cells lack morphological caveolae, but, upon stable transfection of the caveolin1 gene (cav1), form typical flask-shaped caveolae. However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts. Our results demonstrate that the absence of caveolin1 and morphologically identifiable caveolae cannot explain the inability of FRT cells to sort GPI-anchored proteins to the apical domain. Thus, FRT cells may lack additional factors required for apical sorting or for the clustering with GSLs of GPI-anchored proteins, or express factors that inhibit these events. Alternatively, cav1 and caveolae may not be directly involved in these processes.
Lipardi
C
C
Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università degli Studi di Napoli Federico II, 80131 Napoli, Italy.
Mora
R
R
Colomer
V
V
Paladino
S
S
Nitsch
L
L
Rodriguez-Boulan
E
E
Zurzolo
C
C
D000818:Animals; D018958:CD55 Antigens / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure*; D016764:Cell Polarity; D002784:Cholesterol / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D006028:Glycosphingolipids / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D008854:Microscopy, Electron; D016253:Microscopy, Immunoelectron; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014759:Viral Envelope Proteins / Q000378:metabolism
D018958:CD55 Antigens; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D006028:Glycosphingolipids; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1; D002784:Cholesterol